摘要
乳腺癌是女性常见的一种高度异质性的恶性肿瘤。资料表明,目前我国乳腺癌发病率正以每年2%-3%的速度递增,10年间我国主要城市的乳腺癌发病率增长了37%。特别是在30-54岁年龄组中,乳腺癌已成为威胁女性健康的“头号杀手”。受体三阴性乳腺癌(triple-negative breast cancer, TNBC)是近些年学者提出的一个新的乳腺癌亚型,是指肿瘤细胞膜表面缺乏雌激素受体(estrogen receptor, ER)、孕激素受体(progesterone receptor,PR)和人表皮生长因子受体-2(human epidermal growth factor receptor-2,HER-2)的一类乳腺癌。TNBC的发病率占乳腺癌总发病率的11%-20%,尤其好发于年轻女性,且该类乳腺癌具有术后易复发、易远处转移和预后差的特点,对内分泌治疗和针对HER-2的靶向治疗无效,至今仍缺乏令人满意的综合治疗方案。
近年来,非甾体类抗炎药物(nonsteroidal anti-inflammatory drugs,NSAIDs)在抑制肿瘤活性方面展示了很好的应用前景,大量体内外研究显示,常规服用NSAIDs可以大幅度降低乳腺癌等多种肿瘤的发病风险,因此,该类药物引起了广大学者的关注。Harris等研究发现,新一代特异性环氧合酶-2(cyclooxygenase-2,COX-2)抑制剂celecoxib,不仅能明显延迟肿瘤的发生,还可以降低乳腺癌的发病率及肿瘤体积。目前,有学者研究认为,celecoxib的抗肿瘤作用机制可能与降低肿瘤组织COX-2表达、抑制肿瘤细胞的增殖、诱导细胞凋亡有关。但也有学者认为celecoxib的抗肿瘤作用机制并不是依赖抑制COX-2途径,其具体作用机制目前尚存在争议。
Kim和Subhashini等研究报道,celecoxib可通过NF-κB(nuclear factor kappa B, NF-κB)途径抑制宫颈癌、白血病等多种肿瘤细胞的增殖,诱导细胞凋亡。而NF-κB活性失控与肿瘤的形成密切相关,且TNBC中NF-κB异常活化,因此我们想阐明在TNBC中celecoxib是否主要通过下调异常活化的NF-κB而发挥作用。
NF-κB是1986年发现的,从成熟的B淋巴细胞中抽提出的能与免疫球蛋白κ链基因增强子结合的核因子。抑制NF-κB活性可以抑制肿瘤细胞增长、促进细胞凋亡、增加肿瘤细胞对化疗药物的敏感性、抑制炎症反应、减少肿瘤血管生成等。作为NF-κB主要发挥转录调节功能的亚基p65,在肿瘤的发生、发展中发挥着关键性作用,下调p65的表达可以抑制多种肿瘤细胞在体内外的增殖及诱导细胞凋亡。因此,p65的靶向治疗可能在乳腺癌的临床治疗中拥有广泛的应用前景。
目的:本实验中选取三阴性高侵袭性的人乳腺癌细胞系MDA-MB-231作为研究对象,通过不同的实验方法探讨celecoxib的抑癌作用,研究celecoxib在三阴性乳腺癌细胞中对NF-κB活性的影响;检测异常活化的NF-κB信号通路在MDA-MB-231细胞增殖、凋亡以及侵袭中发挥的作用;评价celecoxib是否能够成为TNBC个体化治疗的辅助用药,针对NF-κB信号通路的靶向治疗能否使乳腺癌的化学治疗更加完善,最终为预防、治疗TNBC提供新的治疗思路。
方法:
1以不同浓度的celecoxib作用人三阴性乳腺癌细胞MDA-MB-231,MTT比色法观察其对该细胞增殖的影响,并进一步探讨celecoxib抑制MDA-MB-231细胞的增殖与COX-2途径依赖关系。采用流式细胞仪技术(flow cytometry, FCM)、Hoechst 33258、DNA Ladder形成实验、western blot等方法,研究celecoxib对体外培养的MDA-MB-231细胞凋亡的影响。
2采用伤口愈合实验、transwell侵袭小室实验等,观察celecoxib对MDA-MB-231细胞侵袭行为的影响。并通过RT-PCR法检测肿瘤细胞侵袭转移关键的细胞因子,如血管内皮生长因子(vascular endothelial growth factor, VEGF)、基质金属蛋白酶-2(matrix metalloproteinases-2, MMP-2)及IL-8(interleukin-8, IL-8)的表达变化,酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测IL-8的分泌水平。
3为了研究celecoxib的抗凋亡、抑制细胞侵袭作用与NF-κB信号通路的关系,应用western blot方法检测了不同浓度celecoxib作用后MDA-MB-231细胞的p65、p50、IκBα(inhibitory kappaBα,IκBα)以及磷酸化IκBα(p-IκBα)蛋白的表达情况,并进一步通过western blot法检测p65的核转位变化。采用凝胶电泳迁移率改变分析(electrophoretic mobility shift assay,EMSA)检测用药前后NF-κB DNA结合活性的改变。
4构建表达p65cDNA的真核表达载体,转染MDA-MB-231细胞,观察过表达p65基因对MDA-MB-231细胞增殖、凋亡及侵袭的影响,并通过与celecoxib联合作用,揭示celecoxib和NF-κB信号通路之间的联系。
5利用微小RNA(microRNA,miRNA)技术,构建表达p65miRNA的真核表达载体,沉默p65基因的表达,观察NF-κB信号通路阻断对人乳癌细胞MDA-MB-231增殖、凋亡以及侵袭的影响。
结果:
1 Celecoxib对MDA-MB-231细胞生物学活性的影响
1.1 Celecoxib对MDA-MB-231细胞增殖的影响
MTT分析结果显示,经celecoxib作用24h、48h和72h后,与溶剂对照组相比,对MDA-MB-231细胞增殖有明显的抑制作用(P<0.05),作用效果呈时间、剂量依赖性。Celecoxib作用24h、48h和72h的IC50依次为139.76、68.39、48.27μM。
前列腺素E2(prostaglandin E2,PGE2)是COX-2催化花生四烯酸产生PG的主要产物,细胞分泌PGE2的水平可以用来反映COX-2的活性。不同浓度PGE2与80μM的celecoxib联合作用细胞48h后,与单独应用celecoxib对MDA-MB-231细胞增殖的抑制作用无显著性差异(P>0.05)。提示celecoxib不是通过COX-2和PGE2途径来抑制细胞的增殖。
1.2 Celecoxib对MDA-MB-231细胞凋亡的影响
Celecoxib可以诱导MDA-MB-231细胞凋亡,使部分细胞出现形态不规则、细胞核固缩聚集、空泡样变性、核碎裂等凋亡的形态学变化。Hoechst 33258染色后荧光显微镜下观察,有些细胞核内可见到有浓染致密的荧光颗粒,细胞核呈碎块状、颜色发白。随着celecoxib浓度的增加,凋亡细胞的比例也在逐渐增加,不同浓度组之间有显著差异(P<0.05)。
应用流式细胞技术检测结果显示,80、120μM celecoxib作用48h后,MDA-MB-231细胞凋亡率可达21.08±4.18%、43.51±6.06%,在DNA直方图上出现亚二倍体凋亡细胞峰,与对照组相比差异显著(P<0.05)。说明celecoxib诱导MDA-MB-231细胞凋亡呈现明显的剂量依赖效应。
提取40、80、120μM celecoxib作用72h的MDA-MB-231细胞DNA进行凝胶电泳分析发现,随着celecoxib浓度的增加,DNA片段化更加明显,120μM celecoxib组呈现典型的DNA Ladder图。
Western blot检测结果显示,经40、80、120μM celecoxib作用后,MDA-MB-231细胞呈浓度依赖性使凋亡相关因子caspase-3和聚腺苷酸二磷酸核糖转移酶(poly ADP-ribose polymerase,PARP)表达下调(P<0.05),裂解蛋白表达上调(P<0.05)。
1.3 Celecoxib对MDA-MB-231细胞侵袭性的影响
伤口愈合实验结果显示,celecoxib处理过的细胞向划痕边缘爬行的速度明显减慢,划痕缺损处修复缓慢。随着celecoxib浓度的增加,细胞的运动能力明显下降(P<0.05)。
Transwell侵袭小室实验结果显示,与对照组相比,40、80、120μM celecoxib作用24h后,MDA-MB-231细胞平均穿膜细胞数明显减少(P<0.05)。各剂量组均显示具有抑制细胞穿过人工基底膜的能力,其作用具有剂量-效应相关性。提示celecoxib可以显著抑制MDA-MB-231细胞体外侵袭能力。
RT-PCR检测结果显示,经不同浓度celecoxib处理24h后,MDA-MB-231细胞VEGF、MMP-2及IL-8 mRNA基因的相对表达水平显著低于对照组,差异具有统计学意义(P<0.05)。Celecoxib还可以抑制MDA-MB-231组成性自分泌IL-8(P<0.05),120μM celecoxib可以完全抑制肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)诱导的IL-8分泌效应(P<0.05)。
2 Celecoxib诱导MDA-MB-231细胞凋亡、抑制细胞侵袭的分子机制
第一部分研究证实,celecoxib可以抑制MDA-MB-231细胞增殖、诱导细胞凋亡以及抑制细胞侵袭等生物学行为。本部分研究通过应用不同信号通路抑制剂处理MDA-MB-231细胞,筛选调控该细胞增殖的可能的信号通路。
2.1不同信号通路阻滞剂对MDA-MB-231细胞增殖的影响
MTT实验结果显示,NF-κB抑制剂PDTC(pirrolidine dithiocarbamate,PDTC )和AKT ( protein kinase B/AKT )抑制剂LY294002作用MDA-MB-231细胞24h、48h和72h后,可以抑制细胞的增殖(P<0.05),但PDTC的抑制作用较LY294002的效果更显著。提示AKT和NF-κB信号通路可能是调控MDA-MB-231细胞增殖的主要通路。80μM celecoxib与PDTC、LY294002联用后对细胞的抑制效果更加明显(P<0.05),提示celecoxib可能是对AKT、NF-κB通路均具有抑制作用,从而使增强了每种抑制剂的作用效果。由于Basu等以往的研究证实,celecoxib可以通过抑制AKT信号通路从而抑制该细胞增殖,故我们进一步研究celecoxib是否也通过下调NF-κB信号通路对MDA-MB-231细胞的增殖、凋亡、侵袭等生物学行为发生影响。
2.2 Celecoxib对MDA-MB-231细胞NF-κB p65/p50表达的影响
RT-PCR和western blot结果显示,80μM的celecoxib可以明显降低NF-κB p65的mRNA和蛋白表达水平,与溶剂对照组相比具有显著性差异(P<0.05)。但celecoxib对NF-κB p50的mRNA和蛋白表达水平没有影响(P>0.05)。
2.3 Celecoxib抑制MDA-MB-231细胞NF-κB p65的核转位
Western blot结果显示,80μM celecoxib作用12h后,MDA-MB-231细胞核p65蛋白的表达量降低(P<0.05),提示celecoxib对p65从胞浆至胞核的转位过程具有明显的抑制作用。
2.4 Celecoxib抑制MDA-MB-231细胞IκBα的磷酸化
Western blot结果显示,80μM的celecoxib作用细胞24h后,细胞浆中p-IκBα蛋白表达水平显著下降(P<0.05)。但celecoxib对IκBα总蛋白的表达没有影响(P>0.05)。
2.5 Celecoxib对MDA-MB-231细胞p65与DNA结合能力的影响
EMSA结果显示,在探针冷竞争反应中,正常的标记探针和NF-κB的结合条带会被100倍过量的未标记探针抑制;而在突变探针冷竞争反应中,正常的标记探针和NF-κB结合的条带不会被100倍过量的未标记突变探针抑制。结果提示,MDA-MB-231细胞中NF-κB处于组成性活化状态。
此外,与对照组相比,80μM的celecoxib作用24h后,MDA-MB-231细胞的DNA-蛋白质复合物区带明显变浅,提示celecoxib可以降低MDA-MB-231细胞中NF-κB DNA的结合活性。
2.6 Celecoxib抑制MDA-MB-231细胞中凋亡相关分子Bcl-2的蛋白表达
Western blot分析结果显示,80μM的celecoxib能够明显抑制MDA-MB-231细胞中NF-κB下游Bcl-2分子的表达(P<0.05)。
3外源性p65基因对MDA-MB-231细胞凋亡及侵袭的影响
前两部分的实验研究发现,celecoxib可以抑制p65的表达、核转位及IκBα磷酸化,降低NF-κB DNA结合活性。为了进一步明确celecoxib的作用靶点,我们通过构建p65基因真核表达质粒导入MDA-MB-231细胞,观察外源性p65基因对MDA-MB-231细胞凋亡、侵袭,以及对celecoxib药物作用效果的影响。
3.1真核表达质粒p65cDNA转染MDA-MB-231细胞
将携带有p65基因全长序列的pcDNA3.1质粒瞬时转染MDA-MB-231细胞,RT-PCR检测p65cDNA在转录水平影响靶基因效果。结果显示,p65 mRNA水平较control组和pcDNA3.1空载体组显著增加(P<0.05)。Western blot检测结果显示,瞬时转染p65cDNA后48h,MDA-MB-231细胞p65蛋白表达量较control组和pcDNA3.1空载体组显著增加(P<0.05),与RT-PCR结果的变化规律相一致。
3.2过表达p65基因对MDA-MB-231细胞NF-κB DNA结合活性的影响
EMSA实验结果显示,p65基因的过表达增加了NF-κB复合物结合细胞核内DNA调控元件的能力。
3.3过表达p65基因对MDA-MB-231细胞增殖的影响
过表达p65基因可以促进MDA-MB-231细胞的体外增殖,并可以逆转celecoxib对MDA-MB-231细胞体外增殖的抑制作用。
3.4过表达p65基因对MDA-MB-231细胞周期分布的影响上调p65基因表达后,MDA-MB-231细胞DNA合成的S期细胞比例明显增加(P<0.05),相对静止阶段的G0/G1期细胞比例有所下降。提示过表达p65可以促进细胞由G0/G1期向S期的转化。RT-PCR和western blot结果显示,转染p65cDNA后,细胞的cyclinD1 mRNA水平和蛋白表达水平明显上调,与空载体对照组相比,差异具有统计学意义(P<0.05)。但转染前后细胞的CDK4 mRNA水平和蛋白表达水平,与空载体对照组相比,没有明显的变化(P>0.05)。
3.5过表达p65基因对MDA-MB-231细胞凋亡的影响
FCM检测结果发现,转染p65cDNA的MDA-MB-231细胞给予80μMcelecoxib共孵育48h后,细胞的凋亡率为18.53±2.51%,而空载体对照组细胞为31.21±3.29%,两者比较差异具有统计学意义(P<0.05)。提示p65基因在MDA-MB-231细胞中发挥了抑制细胞凋亡的作用。
Western blot结果显示,转染p65cDNA后,MDA-MB-231细胞的caspase-3、PARP蛋白表达水平明显下降,但未检测到裂解片段,与空载体对照组相比,差异具有统计学意义(P<0.05)。提示过表达p65基因抑制了caspase-3、PARP蛋白的表达和裂解。转染p65cDNA后,MDA-MB-231细胞的Bcl-2蛋白表达水平与对照组相比没有显著的变化(P>0.05)。
3.6过表达p65基因对MDA-MB-231细胞侵袭性的影响
Transwell侵袭小室检测结果显示,转染p65cDNA的MDA-MB-231细胞培养6h后,基本没有细胞侵袭过matrigel胶,而在12h,实验组和对照组侵袭的细胞数无明显差异(P>0.05)。24h后可见转染p65cDNA的细胞克隆侵袭细胞数明显多于对照组,差异具有统计学意义(P<0.05)。
3.7过表达p65基因对MDA-MB-231细胞VEGF、IL-8、MMP-2 mRNA表达的影响
RT-PCR分析结果显示,转染p65cDNA后,MDA-MB-231细胞VEGF、MMP-2和IL-8 mRNA表达水平明显上调,与空载体对照组相比,差异具有统计学意义(P<0.05)。
3.8过表达p65基因对MDA-MB-231细胞分泌IL-8的影响
ELISA结果发现,上调p65基因后,可以促进MDA-MB-231细胞分泌IL-8,与空载体对照组相比,差异具有统计学意义(P<0.05)。
4靶向沉默p65基因对MDA-MB-231细胞凋亡及侵袭的影响
第三部分的实验结果提示,过表达p65基因可以逆转celecoxib诱导MDA-MB-231细胞凋亡,并可促进细胞的侵袭。本部分实验设计了miRNA用来特异性沉默p65基因,观察其对MDA-MB-231细胞生物学行为的影响,探讨其对人三阴性乳腺癌细胞增殖活性及侵袭能力的影响以及可能的临床应用价值。
4.1 p65miRNA真核表达质粒转染MDA-MB-231细胞及效率鉴定
转染p65miRNA质粒48h后,MDA-MB-231细胞内绿色荧光信号较强,转染72h后细胞中绿色荧光强度及出现荧光的细胞数量开始减少,转染效率达70%左右。RT-PCR检测转染效率结果显示,转染24h后,p65mRNA表达量下降,尤其72h时p65 mRNA的表达水平最低,与阴性对照组相比,差异显著(P<0.05)。Western blot检测发现,转染p65miRNA组细胞p65蛋白显著降低,比较差异有显著性(P<0.05)。
4.2沉默p65基因对NF-κB DNA结合活性的影响
EMSA实验结果显示,沉默p65基因后,MDA-MB-231细胞NF-κB DNA-蛋白质复合物区带较对照组明显减弱(P<0.05),说明核蛋白与探针的结合活性显著下降,沉默p65基因降低了NF-κB复合物结合细胞核内DNA调控元件的能力。
4.3沉默p65基因对MDA-MB-231细胞增殖的影响
p65miRNA转染24h、48h及72h后,经MTT检测结果显示, p65miRNA转染组MDA-MB-231细胞出现了生长抑制现象,与对照组相比,差异具有统计学意义(P<0.05)。
4.4沉默p65基因对MDA-MB-231细胞凋亡的影响
FCM检测结果发现,转染p65miRNA 48h、72h后,MDA-MB-231细胞G1峰前面出现亚二倍体峰,细胞凋亡率与阴性对照组比较显著增加,具有统计学意义(P<0.05)。而转染空载体组和转染p65miRNA 24h组未见或仅见微小的亚二倍体峰。
4.5沉默p65基因对MDA-MB-231细胞凋亡相关分子caspase-3、PARP分子表达的影响
Western blot结果显示,转染p65miRNA后,MDA-MB-231细胞caspase-3前体蛋白条带变细,裂解片段条带加深;PARP前体蛋白出现裂解片段。说明下调p65基因表达可以促进caspase-3和PARP前体蛋白表达下降,促进两者的裂解活化。
4.6沉默p65基因对MDA-MB-231细胞IAPs(inhibitors of apoptosis proteins,IAPs)家族分子表达的影响
Western blot结果显示,转染p65miRNA后,MDA-MB-231细胞survivin和XIAP蛋白表达水平,与阴性对照组相比显著下降,差异具有统计学意义(P<0.05)。
4.7沉默p65基因对MDA-MB-231细胞侵袭性的影响
Transwell侵袭小室实验结果显示,与阴性对照组相比,转染p65miRNA的MDA-MB-231细胞平均穿膜细胞数明显减少(P<0.05)。显示p65miRNA具有抑制细胞穿过人工基底膜的能力。
4.8沉默p65基因对MDA-MB-231细胞VEGF、MMP-2、IL-8 mRNA表达的影响
RT-PCR分析结果显示,转染p65miRNA后,MDA-MB-231细胞VEGF、MMP-2和IL-8表达水平明显下降,与空载体对照组相比,差异具有统计学意义(P<0.05)。
4.9沉默p65基因对MDA-MB-231细胞分泌IL-8的影响
ELISA实验结果显示,沉默p65基因后,可以减少MDA-MB-231细胞分泌IL-8的水平,与空载体对照组相比,差异具有统计学意义(P<0.05)。
结论:
1. Celecoxib能够有效地抑制人三阴性乳腺癌细胞MDA-MB-231的增殖,其抑制作用呈浓度及时间依赖性,该作用与COX-2途径无关。Celecoxib可以诱导MDA-MB-231细胞凋亡,作用途径可能与活化caspase-3、PARP分子有关。Celecoxib能够有效地抑制MDA-MB-231细胞的水平运动及侵袭行为,其抑制作用呈剂量-效应依赖性。Celecoxib可以降低MDA-MB-231细胞的VEGF、MMP-2、IL-8 mRNA表达以及IL-8的分泌水平。NF-κB信号通路可能是MDA-MB-231细胞增殖的主要调控通路。Celecoxib可能是通过抑制p65蛋白的合成及其核转位、减少IκB的磷酸化降解,进而抑制NF-κB活性,达到抑癌作用。
2.瞬时转染外源p65基因,可以显著增加MDA-MB-231细胞p65表达水平,过表达p65基因可以增加MDA-MB-231细胞NF-κB DNA的结合活性,促进细胞增殖,逆转celecoxib对细胞活性的抑制作用,可以抑制细胞凋亡,促进细胞的体外侵袭能力,并伴随着VEGF、MMP-2和IL-8 mRNA的表达上调和IL-8分泌增加。
3.转染p65miRNA后,MDA-MB-231细胞生长、增殖能力受到显著抑制,并可以增加凋亡细胞的比例,主要是通过下调survivin和XIAP的表达、裂解活化caspase-3和PARP蛋白而实现的。抑制p65表达可以显著降低MDA-MB-231细胞的体外侵袭能力,主要是通过下调VEGF、MMP-2和IL-8 mRNA的表达,减少IL-8的分泌而实现的。本实验结果为三阴性乳腺癌的分子靶向治疗提供了新靶点。
Objective: Breast cancer is a heterogeneous disease that encompasses several distinct entities with remarkably different biological characteristics and clinical behavior, and threatened to female public health. The incidence of breast cancer is increasing, and current therapy is unable to achieve clinical responses in patients with highly invasive metastatic disease. It is estimated that, in the past decade, Chinese urban cancer registries have documented increased incidence rates of between 20% and 30% for breast cancer. Breast cancer has acclaimed to be the first leading cause of death among 30 to 54 year old women. A new subtype of particular interest is triple-negative breast cancer, namely TNBC, which is characterized by estrogen receptor negative (ER), progesterone receptor negative (PR), and human epidermal growth factor receptor-2 (HER-2) negative using histochemical staining. Although TNBC accounts for 11%-20% of breast cancer cases, it is responsible for a disproportionate number of breast cancer deaths, especially in younger female before menopause. However, there is still absence of suitable treatment for this tumor.
Currently, studies with nonsteroidal anti-inflammatory drugs (NSAIDs) have shown significant effects in reducing the incidence and progression of tumors in both animal models and in treatment of cancer patients. It was therefore of extreme interest when recent epidemiological studies suggested the presence of an inverse association between regular intake of NSAIDs and the relative risk of breast cancer. Celecoxib is a new NSAID that specifically inhibits cyclooxygenase-2 (COX-2). It has significant anti-inflammatory and analgesic properties but lesser side effects than other NSAIDs such as aspirin and ibuprofen, which inhibit both COX-1 and COX-2. Because of emerging evidence suggesting that NSAIDs inhibition of COX reduces the risk of breast cancer, Harris et al conducted a great deal of studies to evaluate the antitumor effects of a specific COX-2 blockade by this compound. Results showed that celecoxib produced striking reductions in the incidence of mammary cancer, tumor burden and tumor volume. Debates have raged continuously for a long period over whether the antitumor effects of celecoxib occur in COX-2-dependent or COX-2-independent manner. However, this mechanism has yet to be clearly defined.
Nuclear factor kappaB (NF-κB) is a pleiotropic transcription factor which was initially found to bind enhancer of immunoglobinκchain extracted from mature B lymphocyte. Regulation of NF-κB activity plays a critical role in tumor development. Inactivation of NF-κB may lead to significant reduction in cancer cell proliferation, tumor angiogenesis, inflammation and enhancement of sensitivity to apoptosis and chemotherapeutics. Preclinical and laboratory data showed that constitutively activated NF-κB was preferentially involved in human breast cancer cell lines, mammary breast cancer tissue sample and carcinogen-induced animal models. Furthermore, highly constitutive NF-κB activation was categorized in the ER negative breast cancer cells compared with those ER positive counterparts, suggesting that NF-κB might become a useful therapeutic target for this subtype of cancer. The family of NF-κB comprises of five members, while p65 subunit contains an extremely active C-terminal transcriptional activation domain (TAD), required for its cellular function. Substantial evidence verified that blocking p65-mediated NF-κB activation could inhibit tumorigenic cancer cell line proliferation and promote apoptosis. Thus, these results showed that incapacitating NF-κB mediated by silencing p65 prevents xenograft tumor growth of cells that otherwise readily form tumors in recipient animals, suggesting that silencing p65 may have promising perspective in tumor clinical therapy.
Methods: in the present studies, triple-negative and highly invasive breast cancer cell line MDA-MB-231 was selected as study object. Multiple experimental methods, such as flow cytometry (FCM), western blot, electrophoretic mobility shift assay (EMSA), were employed to investigate the possible antitumor mechanisms of celecoxib, and screen its potential signaling transduction pathway. To evaluate the underlying roles of constitutively activated NF-κB play in proliferation, apoptosis and invasion of MDA-MB-231 cells. Finally, our aim is to verify whether celecoxib could become an assistant candidate in synergy with chemotherapy to cure TNBC, and to inactivate NF-κB by interfering p65 could provide an additional target therapy to complement antineoplastic dosages of traditional medicine.
Results:
1 The effect of celecoxib on MDA-MB-231 cell biological behaviour
In the initial part, we investigated the influential effect of celecoxib on proliferation, apoptosis and invasiveness behavior of MDA-MB-231 cells. The results were as follows:
1.1 Celecoxib inhibits MDA-MB-231 cell proliferation
Celecoxib at concentrations of 40, 80 and 120μM was used to treat MDA-MB-231 cell line for 24h, 48h and 72h,respectively. The rate of proliferation in response to celecoxib treatment was analyzed by measuring MTT assay. Significant inhibition of proliferation was observed in MDA-MB-231 cells in a dose- and time-dependent manner (P<0.05). The IC50 of celecoxib at 24h, 48h and 72h was 139.76、68.39、48.27μM.
Prostaglandin E2 (PGE2) is the major prostaglandin that COX-2 derived arachidonic acid into, which can be used to determine the COX-2 activity in quantity. To determine whether celecoxib-induced growth inhibition could be reversed by exogenous PGE2, we added PGE2 to the culture of MDA-MB-231 cells treated with constant dose (80μM) of celecoxib for 48h. In MDA-MB-231 cells, growth inhibition induced by 80μM celecoxib could not be restored by addition of exogenous PGE2, thereby suggesting that celecoxib suppressed MDA-MB-231 cell growth was independent of PGE2 levels and COX-2 pathway (P>0.05).
1.2 Celecoxib induces MDA-MB-231 cell apoptosis
To investigate whether the observed growth inhibition mediated by celecoxib was associated with induction of programmed cell death. We used Hoechst 33258 staining, FCM analysis and DNA fragmentation assay to observe nuclear fragmentation, and monitor the changes of cells after celecoxib treatment. Under the phase contrast microscope, cells exhibited a dramatic morphologic change after 48h of drug treatment. Celecoxib at concentrations of 80μM and 120μM caused significant increase in the percentage apoptotic cells (21.08±4.18%, 43.51±6.06%, P<0.05, respectively). It is showed that celecoxib treatment results in the formation of DNA fragments in MDA-MB-231 cells, as determined by agarose gel electrophoresis at 72h. As the intrinsic mitochondrial apoptotic pathway is relatively more important than the death receptor pathway for the induction of apoptosis by chemotherapeutic drugs, we examined the executioners, caspase-3 and poly-ADP-ribose polymerase (PARP) by immunoblotting. Results showed that in celecoxib-treated cells, they were both cleaved into their specific active forms. The effector caspases-3 proteolytically cleaves and activates several other caspases as well as several other apoptotic proteins including PARP, which is rapidly activated during the cellular response to DNA damage, and is a part of safeguard mechanisms protecting cells from genotoxic damage. These results suggested that celecoxib-induced apoptosis in MDA-MB-231 cells is due to activation of caspases-3 and its subsequent cleaved apoptotic protein PARP.
1.3 Celecoxib blocks MDA-MB-231 cell migration and invasion
Cancer cell migration and invasion play very important roles in cancer metastasis. Consequently, we further studied the effects of celecoxib on migration and invasion as well as the related angiogenic factors, such as vascular endothelial growth factor (VEGF), matrix metalloproteinases-2 (MMP-2) and interleukin-8 (IL-8) mRNA expression of MDA-MB-231 cells.
In vitro, wound healing assay results showed that the migration of MDA-MB-231 mock cells and celecoxib-treated cells differ at 24h after wounding with mock cells migrating almost twice as slowly as 80μM celecoxib-treated cells (P<0.05). These data suggested that celecoxib could affect MDA-MB-231 cells on horizontal migratory activity induced by wound, which was consistent with the prognostic value of celecoxib in tumoural disease and metastatic potential.
Next, we examined the motility of different concentration celecoxib-treated cells with transwell cell invasion assay. Results revealed that with the presence of 120μM celecoxib significantly reduced the invasion of MDA-MB-231 cells (P<0.05). The level of celecoxib-induced cell invasion was significantly reduced by 67% of control level (P<0.05). These experiments indicated that treatment of celecoxib in MDA-MB-231 cells inhibits cell motility and invasion activity in vitro.
The expression of VEGF, MMP-2 and IL-8 mRNA in MDA-MB-231 cells was examined by RT-PCR analysis. As shown, all cells expressed the same level ofβ-actin without difference (P>0.05), but celecoxib could significantly lower these cytokines mRNA expression (P<0.05).
The secretion of IL-8 was further confirmed by enzyme-linked immunosorbent assay (ELISA). The expression level of celecoxib-treated cells was significantly lower than those in control groups (P<0.05). Besides, celecoxib could abrogate the potential of TNF-α-induced IL-8 secretion (P<0.05).
2 Study on the underlying mechanism of celecoxib inhibiting MDA-MB-231 cell growth, invasion and promoting apoptosis
In previous study, we knew that celecoxib had strong inhibition on MDA-MB-231 cell growth and invasion, and could induce cell apoptosis. To explore the underlying mechanism of these phenomena, various pathway signaling inhibitors were employed to screen its possible targets.
2.1 Various signaling pathway inhibitors to screen the potential target of celecoxib
Signaling pathway inhibitors including SP600125 (JNK inhibitor, Jun N-terminal kinase inhibitor), PD98059 (ERK inhibitor, extracellular signal-regulated kinase inhibitor), SB203580 (p38 inhibitor), PDTC (pirrolidine dithiocarbamate, NF-κB inhibitor) and LY294002 (AKT inhibitor, protein kinase B/AKT inhibitor) were employed to treat cells alone or in combination with celecoxib. MTT assay results showed that PDTC and LY294002 could cause inhibition of MDA-MB-231 cell proliferation (P<0.05). Especially, combinations of PDTC and celecoxib at different concentrations showed a strong synergistic effect on cell proliferation. Basu et al reported that the mechanism of celecoxib-induced growth arrest was by induction of apoptosis, associated with reduced activation of AKT. Hence, we want to figure out whether NF-κB aberrant activation played a crucial part in highly invasive breast carcinoma cell line MDA-MB-231, and whether celecoxib exerted its antitumor effect through inactivation of NF-κB.
2.2 Celecoxib inhibits NF-κB p65 expression but not p50
Unstimulated MDA-MB-231 cells demonstrated somewhat higher levels of constitutive NF-κB p65 and p50 expression. However, treatment with 80μM celecoxib resulted in additional reduction in p65 expression (P<0.05), but no influence on p50 expression (P>0.05).
2.3 Celecoxib inhibits NF-κB p65 nuclear translocation
Cytoplasmic p65 level was slightly decreased after celecoxib stimulation. In contrast, celecoxib significantly suppressed p65 nuclear translocation after 24h treatment (P<0.05). The data clearly demonstrated that celecoxib could influence the process of p65 from cytoplasm to nucleus.
2.4 Celecoxib inhibits IκBαphosphorylation
Phosphorylation and degradation of IκBαis the initiating event in NF-κB activation and translocation, and upregulation of IκBαexpression constrains p65/p50 to the cytosol and inhibits in?ammatory NF-κB activity. Accordingly, we also tested the hypothesis that celecoxib acted on p65 localization via effects on IκBαprotein expression. Results showed that celecoxib inhibited IκBαphosphorylation, indicating that intact IκBαcould suppress p65 translocation (P<0.05).
2.5 Celecoxib causes inactivation of NF-κB DNA Binding activity
To investigate whether celecoxib modulated NF-κB DNA binding activity in MDA-MB-231 breast cancer cells, EMSA was performed. Autoradiography revealed that NF-κB DNA Binding activity was constitutively activated in unstimulated cells. However, NF-κB DNA binding activity was decreased in cells treated with celecoxib for 24h. Besides, in competition assay, mutant oligonucleotide failed to compete with the specific binding, while unlabeled specific oligonucleotide counteracted the effects of the labeled. The above results indicated that the induction of apoptosis by celecoxib may be potentially mediated by the downregulation of NF-κB transcription factor activity which, in turn, may be responsible for both cell growth inhibition and induction of apoptotic processe.
2.6 Celecoxib inhibits the expression levels of Bcl-2
To explore the possible role of Bcl-2 family members in celecoxib -induced apoptosis, the effects of celecoxib on the expression level of Bcl-2 by western blot analysis was examined. Exposure of cells to 80μM of celecoxib resulted in downregulation of Bcl-2 significantly (P<0.05).
3 Study on the effect of overexpression p65 on cell apoptosis and invasion in MDA-MB-231 cells in vitro
Previous studies showed that celecoxib could inhibit p65 expression, translocation and IκBαphosphorylation, therefore inactivation of NF-κB DNA binding activity. We hypothesized that NF-κB p65 signaling in breast cancer cells MDA-MB-231 could be a selective target of celecoxib treatment. In this part, we conducted p65cDNA transfection into MDA-MB-231 cells and observed the effects of endogenous p65 on cell apoptosis, invasion and its potential influence on celecoxib treatment.
3.1 NF-κB p65cDNA transfection
The recombinant plasmid, designated as pcDNA3.1-p65, eukaryotic expression plasmid encoding p65 gene coding region was constructed. The recombinant p65cDNA transfected into breast cancer cells MDA-MB-231 by the vector of lipofectamine 2000. Then total cell RNA, the cytoplasmic proteins and nuclear proteins were extracted. NF-κB p65 mRNA and protein expression were detected by RT-PCR and western blot analyses, respectively. Results showed that MDA-MB-231 cells which have been transfected p65cDNA recombination plasmids could significantly enhance the mRNA and protein level of p65 gene expression (P<0.05).
3.2 p65cDNA transfection enhanced NF-κB DNA binding activity
Importantly, EMSA results showed that NF-κB p65cDNA transfection enhanced the NF-κB DNA binding activity significantly in vitro (P<0.05).
3.3 p65cDNA transfection promotes cell growth
We applied MTT to detect the proliferation capability of MDA-MB-231 cells after transfection. The results suggested the growth and proliferation of MDA-MB-231 cells transfected with the recombinant of p65cDNA were promoted than those not transfected. Moreover, the growth and proliferation of MDA-MB-231 cells transfected with the recombinant of p65cDNA in combination of celecoxib treatment were faster than those without transfection.
3.4 p65cDNA transfection promotes S phase progression of MDA-MB-231 cells
FCM was done to further define the mechanism by which p65cDNA transfection promoted the growth of MDA-MB-231 cells. p65cDNA transfection led to an increase in the percentage of cells in S and a corresponding decrease in the percentage of cells in G0/G1 phase (P<0.05). Results showed that overexpression p65 gene might alter the progression to S phase of MDA-MB-231 cells.
Cyclin D1 is involved in the early- to mid- G1 phase of the cell cycle in association with its catalytic partner CDK4. Emerging evidence indicated that cyclin D1 was essential for G1 progression in this breast cancer cell line. Our results showed that overexpression p65 gene could increase both cyclinD1 mRNA and protein expression (P<0.05), but little effects on CDK4 (P>0.05).
3.5 p65cDNA transfection inhibits cell apoptosis
FCM results revealed that the rate of apoptosis decreased in p65cDNA transfected MDA-MB-231 cells compared with control cells after 80μM celecoxib treatment. It indicated that the constructed recombination plasmids can suppress the apoptosis effect of MDA-MB-231 cells induced by celecoxib. Using western blot analysis, we observed that celecoxib downregulated the expression of caspase-3 and PARP in NF-κB p65cDNA-transfected or parental breast cancer cells (P<0.05). However, there was little change found in Bcl-2 expression (P>0.05). These results are consistent with the cell growth inhibition assay, suggesting that greater cell growth inhibition resulting from the celecoxib treatment is partly mediated through the induction of greater apoptosis in breast cancer cells. These results provide mechanistic support in favor of our claim that the apoptosis-inducing effect of celecoxib is partly mediated through the NF-κB pathway.
3.6 p65cDNA transfection promotes cell invasion
The result from transwell cell invasion assay indicated that p65cDNA transfection significantly promoted the invasion of MDA-MB-231 cells, as compared with mock-transfected and control vector–transfected cells (P<0.05). Furthermore, the mRNA levels of VEGF、IL-8 and MMP-2 mRNA were showed great enhancement in MDA-MB-231 cells transfected p65cDNA (P<0.05). MDA-MB-231 cells constitutively expressed a high level of IL-8, however, p65cDNA transfection can significantly accelerate the secretion (P<0.05).
4 Study on the effect of p65miRNA by targeting p65 gene on cell apoptosis and invasion in MDA-MB-231 cells in vitro
In the third part, overexpression p65 could abrogate the ability of celecoxib-induced cell apoptosis and promote the invasiveness and metastasis of MDA-MB-231 cells. Accordingly, we investigated the down-modulation of p65 expression and function by artificial microRNA (miRNA) by measuring mRNA and protein levels in the breast cancer cell line MDA-MB-231, which has a high level of p65 expression.
4.1 NF-κB p65miRNA transfection
Primarily, the recombinant expressive plasmid p65miRNA in pcDNATM6.2-GW/EmGFP-miR-based miRNA with a pre-microRNA sequence was constructed and transfected to MDA-MB-231 cells. Fluorescene microscope was applied to observe green fluorescence, which suggested cells transfected successfully. RT-PCR and western blot analyses were used to detect the expression of p65 at mRNA and protein level. The results showed that the MDA-MB-231 cells which have been transfected the p65miRNA recombination plasmids could significantly inhibit the mRNA and protein level of p65 expression (P<0.05).
4.2 NF-κB p65miRNA transfection reduced NF-κB DNA binding activity
EMSA results clearly demonstrated that NF-κB p65miRNA transfection decreased the NF-κB DNA binding activity significantly (P<0.05), suggesting that silencing p65 gene led to inactivation of NF-κB.
4.3 Silencing p65 inhibits MDA-MB-231 cell growth
MTT results showed that p65miRNA transfection inhibited cell proliferation of MDA-MB-231 cells in vitro as compared with mock-transfected and control vector–transfected cells (P<0.05).
4.4 Silencing p65 sensitizes MDA-MB-231 cells apoptosis
FCM results showed that rate of apoptosis increased in cells transfected with the recombinant of p65miRNA than those not transfected. To examine whether silencing p65 promoted caspase activation, we used western blot to detect the changes of caspase-3 and PARP. In consistent with previous data, p65miRNA transfection profoundly reduced the level of precatalytic caspase-3 and PARP (P<0.05). To determine the definite mechanisms by which targeting p65 promoted apoptosis, we examined the effect of p65miRNA transfection on IAPs gene expression. Results revealed that downregulation p65 could lead to reduction in survivin and XIAP protein level as well (P<0.05).
4.5 Silencing p65 inhibits MDA-MB-231 cells migration and invasion
Transwell cell invasion assay was used to determine invasion capability change of breast cancer cells transfected p65miRNA. Our results demonstrated that lowering levels of p65 mRNA and protein inhibited cell invasion considerably (P<0.05). Our previous investigation has demonstrated that p65 overexperssion could lead to upregulate angiogenic cytokines, which were related to tumor metastasis and angiogenesis. Herein, to verify whether VEGF, MMP-2 and IL-8 were blocked due to silence of p65 expression with this artificial miRNA, RT-PCR for detecting of mRNA was performed. Results revealed that VEGF, MMP-2 and IL-8 were downregulated in the breast cancer cell line MDA-MB-231 transfected with p65miRNA after 24h (P<0.05). Control-miRNA-transfected MDA-MB-231 cells still expressed the same levels. Besides, ELISA assay showed that IL-8 secretion was reduced significantly after blocking p65 expression (P<0.05).
Conclusions:
1. Celecoxib inhibits MDA-MB-231 cell proliferation in a dose- and time-dependent manner, whereas probably independent of COX-2 pathway. Celecoxib induces apoptosis in MDA-MB-231 cells by increasing DNA ladder formation and the activity of caspase-3 and PARP. The mechanism may be involved in inhibition of the expression, nuclear translocation of p65 and IκBαphosphorylation, which lead to inactivation of NF-κB DNA binding activity and reduction its downstream Bcl-2 expression. Furthermore, celecoxib could suppress the migration and invasion of MDA-MB-231 cells, the mechanism of which might be concerned with downregulation of VEGF, MMP-2 and IL-8 mRNA levels, and reduction in IL-8 secretion.
2. Overexpression of p65 promotes p65 expression in both mRNA and protein levels, and results in enhancement of NF-κB DNA binding activity. p65cDNA transfection abrogates the rate of apoptosis induced by celecoxib and promotes the invasiveness ability of MDA-MB-231 cells. Overexpression of p65 overrode caspase-3 and PARP activation, whereas no influence on Bcl-2 expression. Besides, VEGF, MMP-2 and IL-8 mRNA levels plus IL-8 secretion were upregulated caused by p65cDNA transfection.
3. Reduction of endogenous p65 by miRNA treatment significantly impaired NF-κB activation, induced apoptosis, and reduced invasion of MDA-MB-231 cells. The mechanism may be related to activation of caspase-3 and PARP. p65miRNA transfection significantly reduced the levels of XIAP and survivin compared with control. Furthermore, p65miRNA suppressed invasive ability of MDA-MB-231 cells, in level with VEGF, MMP-2 and IL-8 downregulation, and reduced IL-8 secretion.
引文
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